As described in Colman et al., Editors, Hemostasis and Thrombosis, Third Edition, J.B. Lippincott Company, Philadelphia, 1994, pages 33-36, 62-63 and 94-105, human Factor IX is a 415 amino acid glycoprotein (Mr.apprxeq.57,000, 17% carbohydrate). Factor IX is a proenzyme that has no catalytic activity. During the coagulation cascade, it is cleaved by Factor XIa to produce catalytically active Factor IXa. A wide variety of Factor IX gene mutations are found in patients with hemophilia B. Among these are mutations in the enzyme active site, including a Ser365 to Arg mutation and mutations near His221. (Colman et al., page 63) These mutations affect the ability of the active site to proteolytically cleave its Factor X substrate. Mutations of Gly363 to Val were found to be functionally normal but unable to activate Factor X (Colman et al., page 104).
The gene for Factor IX has been identified, cDNA for Factor IX has been isolated, sequenced, and cloned into expression vectors, and recombinant Factor IX has been expressed. See, for example, Durachi et al., "Isolation and characterization of a cDNA coding for human Factor IX," Proc. Natl. Acad. Sci. USA 79: 6461, 1982 (GenBank Accession Nos. J00136 and 182690); Choo et al., "Molecular cloning of the gene for human anti-haemophilic factor Factor IX," Nature 299: 178, 1982; Anson et al., "the gene structure of human anti-haemophilic factor Factor IX," EMBO J. 3:1053, 1984; Yshitake et al., "Nucleotide Sequence of the gene for human Factor IX," Biochemistry 24:3736, 1985 (GenBank Accession No. 182,613); Anson et al., "Expression of active human clotting Factor IX from recombinant DNA clones in mammalian cells," Nature 315:683,1985; Busby et al., "Expression of active human Factor IX in transfected cells," Nature 316:271, 1985; de la Salle et al., "Active gamma carboxylated human Factor IX expressed using recombinant DNA techniques," Nature 316: 268, 1985; and Kaufman et al., "Expression, purification, and characterization of recombinant gamma-carboxylated factor IX synthesized in Chinese hamster ovary cells," J. Biol. Chem. 261:9622, 1986. See also Brownlee et al., UK Patent Application GB 2 125 409 A, published Mar. 7, 1984; Anson et al., U.S. Pat. No. 5,171,569, issued Dec. 15, 1992; Muelien U.S. Pat. No. 5,521,070, issued May 284 1996; Kaufman et al., U.S. Pat. No. 4,770,999, issued Sep. 13, 1988; and Barr et al., U.S. Pat. No. 5,460,950, issued Oct. 24, 1995.
In addition, Benedict et al. (1994) Texas Heart Institute Journal Vol 21, No. 1, pp 85-90 disclose that infusion of Factor IXai at concentrations sufficient to inhibit intravenous coagulation did not produce bleeding significantly different from that in control animals. Therefore, the invention disclosed herein was unexpected in view of this report.